This invention relates to enzymatic assays, and more particularly to enhancing the reactivity of peroxidase for use in catalyzed reporter deposition.
Peroxidase, because of its high turnover rate, good stability, and availability is widely used in enzyme-based analytical methods. For example, horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of a large variety of hydrogen-donating substrates with hydrogen peroxidase. HRP is also one of the preferred enzymes for use in catalyzed reporter deposition.
Catalyzed reporter deposition (CARD) is a novel method of signal amplification which constitutes the subject matter of U.S. Pat. Nos. 5,863,748; 5,688,966; 5,767,287; 5,731,158; 5,583,001 and 5,196,306 all of which are incorporated herein by reference. It is also discussed in Bobrow et al., Journal of Immunological Methods, 125: 279-285 (1989) and in Bobrow et al., Journal of Immunological Methods, 137: 103-112 (1991).
The method utilizes an analyte-dependent enzyme activation system (xe2x80x9cADEASxe2x80x9d) to catalyze the deposition of a detectable label onto the solid phase of an assay platform. These enzymatically deposited labels may be detected directly or indirectly and results in signal amplification and improved detection limits. In a preferred embodiment, HRP is the enzyme.
HRP reacts with a conjugate consisting of a detectably labeled substrate specific for the ADEAS. When the ADEAS and the conjugate react, an activated conjugate is formed which deposits covalently wherever receptor site for the activated conjugate is immobilized.
For analytical use, substrate oxidation by HRP has been used to generate products which become colored, fluorescent or chemiluminescent. These products either remain soluble or become insoluble and precipitate on the solid phase. The CARD method differs in this respect as the products of the detectably labeled phenol substrate become covalently bound to the solid phase.
To improve detection limits in analytical methods, it is desirous to increase or enhance the substrate to product conversion by enzymes. Although a substance which enhances HRP catalysis regardless of the substrate used has not been discovered, several enhancers specific for HRP substrates which form soluble products have been described. One enhancer specific for the substrate diaminobenzidine, which forms an insoluble product has been described. Enhancers for substrates which, by the catalytic activity of HRP, form covalently depositable products have not been described.
J. R. Whitaker and A. L. Tappel, Biochimica et Biophysica Acta, pages 310-317, Vol. 62, 1962 show that KCl, NaCl, Na2SO4 and to a lesser extent, LiCl enhance the oxidation of guaiacol.
U.S. Pat. No. 4,598,044 issued to Kricka et al. on Jul. 1, 1986 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, which forms a soluble chemiluminescent product, by various phenolic compounds.
U.S. Pat. No. 4,729,950 issued to Kricka et al. on Mar. 8, 1988 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, by various aromatic amine compounds. Tables 1 and 2 summarize various substrate/enhancer combinations. The Tables and the discussion (column 3 line 67 to column 4 line 34) lead to the conclusion that whether an HRP catalyzed oxidation of a substrate will be enhanced by a given compound is not predictable.
U.S. Pat. No. 5,629,168 issued to Kricka on May 13, 1997 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, by aromatic organoboron compounds.
U.S. Pat. No. 4,521,511 issued to Stout on Jun. 4, 1985 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,2xe2x80x2-azino-di(3-ethyl-benzothiazolone-6-sulfonic acid), by various phenolic compounds.
W. Straus, Journal of Histochemistry and Cytochemistry, Vol. 30, pages 491-493, 1982, shows that imidazole enhances the HRP catalyzed oxidation of diaminobenzidine which forms in insoluble product.
A. S. H. de Jong et al., Histochemical Journal, Vol. 17, pages 1119-1130, 1985 also show that imidazole enhances the oxidation of diaminobenzidine by approximately four fold, a substrate combination of p-phenylenediamine-pyrocatechol by two fold and has no effect on the substrate 4-chloro-1-naphthol, all of which form insoluble products.
The aforementioned enhancers, with the exception of imidazole, only enhance the conversion of soluble substrates to soluble products. In addition, the enhancers are substrate specific. The KCl, NaCl, Na2SO4 and LiCl enhancement of the oxidation of guaiacol is specific for guaiacol. These salts do not enhance the oxidation of substrates which form insoluble products nor do they enhance the oxidation of commonly used substrates that form soluble products, such as orthophenylediamine or tetramethylbenzidine. The enhancers for 2,3-dihydro-1,4-phthalazinedione also do not enhance the oxidation of substrates which form insoluble products nor do they enhance the oxidation of commonly used substrates that form soluble products, such as orthophenylediamine or tetramethylbenzidine. Imidazole, which has been demonstrated to enhance the oxidization of diaminobenzidine, has a marginal effect on p-phenylenediamine-pyrocatechol, no effect on 4-chloro-1-naphthol, and no effect on substrates which form covalently depositable products. Whether the oxidation of a given substrate by HRP will be enhanced by a given compound cannot be predicted.
Accordingly, it would be advantageous and desirable to have reagents for enhancing the catalysis of HRP and to have an enhancement effect greater than would be expected based on previous technology.
The present invention concerns enhancing the catalysis of HRP in a Catalyzed Reporter Deposition (CARD) method by reacting a conjugate comprising a detectably labeled phenol with a peroxidase enzyme, wherein the reaction is carried out in the presence of an enhancing reagent including at least one inorganic salt, an organic enhancing compound or mixtures of both the inorganic salt and the organic enhancing reagents of the structure, 
wherein when X is B(OH)2, Y is I; or wherein when X is OH, Y is a halogen, or Q-R, wherein Q is a linear or branched 1-12 heteroatom alkyl chain, wherein the heteroatoms can be selected from C, N, O and S, wherein the bonds connecting the alkyl chain are single or double, wherein any carbon in the alkyl chain optionally includes a substituent selected from xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, and xe2x80x94SH, and wherein R is selected from xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, and xe2x80x94CH3.